Introduction
Indeterminate biliary strictures (IBS) still pose a major challenge in endoscopic
diagnostics today [1]
[2]. Endoscopic retrograde cholangiopancreatography (ERCP) represents the standard procedure
to obtain cytological/histopathological material within the bile duct [3]. Brush cytology and fluoroscopic guided forceps biopsy are routinely used for this
purpose [4]. However, inadequate sensitivity, as low as 45 % is frequently reported for both
methods [2].
Hence, different approaches to obtain tissue are being developed. Per oral cholangioscopy
allows direct visualization of the biliary tract and targeted biopsies of the suspected
area. However, recent studies still show moderate sensitivity [5]. Endoscopic ultrasound-guided fine-needle aspiration is also discussed as an uprising
diagnostic modality with a varying spread of sensitivity [6]. Due to these insufficient outcomes, alternative methods are necessary to increase
the sensitivity and diagnostic rate.
The cryobiopsy technique is a new method for tissue extraction which is already used
in pneumology in clinical routine [7]. Its principle is based on the Joule-Thomson Effect, which causes a sudden cooling
effect in the cryoprobes tip with an immediate attachment to the surrounding tissue
by freezing to it [8]
[9]. The cryoprobe can subsequently be extracted with the attached tissue.
The purpose of this ex vivo clinical study was to investigate the feasibility and
tissue quality of cryobiopsy in comparison to standard biopsy forceps for retrieval
of native and pathologically altered bile duct tissue.
Patients and methods
This prospective study was approved by the Ethics Committee of the University Tuebingen
(No. 495/2017BO2). Patients with suspected tumor or confirmed malignant obstruction
of the common bile duct (CBD) or in the pancreatic region who underwent a pancreaticoduodenectomy
were included. For each patient, written informed consent for participation in the
study and for data protection was obtained before intervention.
A cryoprobe prototype (ERBE Elektromedizin GmbH, Tuebingen, Germany) with 160 cm in
length and 1.1 mm in diameter was used for cryobiopsy. The cryoprobe was connected
to a cryo device (ERBECRYO2, ERBE Elektromedizin GmbH, Germany) serving as gas supplier
using carbon dioxide as cooling agent ([Fig. 1]).
Fig. 1 Representative picture of cryo device and cryoprobe (tip of the probe shown in magnified
view)
Two different forcepes were used as control to perform biopsies; a standard cholangioscopic
forceps (Medwork Endobite, Medwork GmbH, Hoechstadt an der Aisch, Germany) with a
closed diameter of 1.8 mm and a standard gastric biopsy forceps (Boston Scientific
Radial Jaw 4, Marlborough, Massachusetts, United States) with a closed diameter of
2.2 mm.
The explanted tissue contained parts of the pancreas, CBD, and duodenum. The CBD was
incised lengthwise and fixated with tweezers onto an underlayer to visualize the designated
biopsy area. Either cryobiopsy or forceps biopsy was conducted. When cryobiopsy was
performed, the probe was placed tangentially onto the incised bile duct and tissue
could be retrieved with a quick pull on the probe. Activation time for cryobiopsy
was varied between 1 and 6 seconds in initial experiments to optimize tissue retrieval.
An activation time of three seconds was used for the majority of biopsies ensuring
best specimen quality while causing least tissue damage. Obtained tissue samples were
fixed in formalin, embedded in paraffin and subsequently stained in hematoxylin and
eosin. The samples were analyzed and assessed by two experienced pathologists who
were blinded to the biopsy method. The primary aim was to evaluate the specimen quality
described by a histological assessment score using a 7-point Likert scale (see Supplementary Table 1) as has been also described previously [10]. Data were also collected and analyzed for feasibility (a biopsy attempt was feasible,
if any specimen was obtained), specimen area (mm2) and representativity. Representativity was assessed qualitatively as the presence
of evaluable bile duct epithelium to rule out malignancy or, in case of carcinoma,
the presence of ample tumor cells and possible invasive growth allowing a definite
diagnosis of malignancy. Discrepancies in the judgment of representativity were solved
by case discussion between both pathologists. The sample area was determined by rendering
the fixed tissue and automatically calculating the surface using the ZEN blue Edition
software tool (Carl Zeiss Microimaging Goettingen, Germany).
Statistical analysis
All data were analyzed with Graph Pad Prism Version 8 (GraphPad Software, LCC) or
R (R Core Team [2020]), using the tidyverse package, version 1.3.0. To calculate statistical
significance, Mann-Whitney test, Wilcoxon test or Chi-square test were applied, depending
on data type. Interobserver reliability for representativity was expressed by Cohen's
kappa coefficient. All p-values are two-sided and P < 0.05 was considered as statistically significant. To correct for multiple comparisons,
the Bonferroni–Holm method was applied. A post hoc power analysis was performed deriving
from the current study data. Effect size was calculated where comparable biopsy pairs
were available (ten pairings of cryobiopsy vs. gastric biopsy forceps). As effect
measure, the difference in mean size of the histologic specimens retrieved by each
method in each patient was used. Power calculation was performed with the power.t.test
function in R. The mean paired difference between size of cryobiopsy and gastric biopsy
forceps was 3.2 mm2, with a standard deviation of 4.8. With n = 10 pairs, a delta of 0.667 and a significance
level of 0.05, the power was approximately 47 %. Because this was not a randomized
study a post hoc analysis was performed to detect bias in the chronological order
of biopsy attempts. Every biopsy attempt per patient was ranked according to its chronological
order and mean ranks per patient and instrument were calculated. Median rank for attempts
compared in the analysis with the gastric biopsy forceps was 5.0, while cryobiopsy
attempts had median rank of 4.0 (P = 0.089). Therefore, although the distribution of biopsy attempts was not completely
equal between patients and biopsy technique, there was likely no statistical bias
introduced.
Results
Population
A total of 14 patients (9 male/5 female) with a mean age of 70.8 years (range: 50
to 84) were included in this study. Final histologic assessment confirmed pancreatic
cancer for eight patients, two had cholangiocellular carcinoma, one was diagnosed
with ampullary adenocarcinoma, one with chronic pancreatitis, one with neuroendocrine
tumor, and one with cystadenoma without any proof of dysplasia. Clinical patient data
are summarized in [Table 1].
Table 1
Clinical data from patients.
|
Patient
|
Gender
|
Age at resection
|
Histology/tumor
|
Biopsy attempts successful and overall taken biopsies
|
|
1
|
Female
|
69
|
Pancreatic carcinoma
|
3/3
|
|
2
|
Female
|
79
|
Pancreatic carcinoma
|
0/6
|
|
3
|
Male
|
61
|
Pancreatic carcinoma
|
4/9
|
|
4
|
Female
|
77
|
Pancreatic carcinoma
|
3/6
|
|
5
|
Male
|
76
|
Pancreatic carcinoma
|
5/8
|
|
6
|
Female
|
56
|
Cholangiocellular carcinoma
|
4/7
|
|
7
|
Male
|
76
|
Pancreatic carcinoma
|
4/7
|
|
8
|
Male
|
83
|
Pancreatic carcinoma
|
5/10
|
|
9
|
Male
|
64
|
Ampullary adenocarcinoma
|
4/10
|
|
10
|
Female
|
50
|
Chronic pancreatitis
|
6/9
|
|
11
|
Male
|
60
|
Neuroendocrine tumor
|
5/9
|
|
12
|
Male
|
82
|
Pancreatic carcinoma
|
2/9
|
|
13
|
Male
|
84
|
Cholangiocellular carcinoma
|
9/9
|
|
14
|
Male
|
74
|
Cystadenoma
|
3/10
|
Feasibility of ex vivo biopsies
A total of 112 biopsy attempts with either cryobiopsy, cholangioscopic forceps or
gastric biopsy forceps were evaluated ([Fig. 2a]). Fifty-seven (50.9 %) of these were successful, meaning tissue particles could
be retrieved from the bile duct ([Table 2]). Another ten tissue samples were excluded from the analysis of size and assessability
due to a lack of histopathological evaluable material (either technical error or missing
evaluable bile ducts within the specimen, [Fig. 2a]). Attempts with cholangioscopic forceps showed an especially low success rate (20.7 %
[6/29]). Therefore, gastric biopsy forceps (success rate of 69.7 % [23/33]) was additionally
performed as an alternative biopsy modality to allow a meaningful comparison. There
was no statistically significant difference between the success rate of cryobiopsy
(56 % [28/50]) and gastric biopsy forceps (P = 0.31). Because only three cholangioscopic forceps biopsies from a single patient
could be fully evaluated, this modality was excluded from further comparisons.
Fig. 2 a Number of biopsy attempts for each instrument (no tissue retrieved, red; evaluable
for all subsequent analyses, green; technical error during processing, blue; no evaluable
bile ducts within the histopathological specimen, purple). b Measured areas of tissue samples (cryobiopsy vs. gastric biopsy forceps). Scatter
blots including individual datapoints and boxes displaying medians, 25th and 75th
percentiles, and whiskers extending to 1.5 of interquartile range. c Histological assessment by two independent histopathologists according to the histopathological
assessability score (evaluation see Supplementary Table 1, cryobiopsy vs. gastric biopsy forceps). Scatter blots including individual datapoints
and boxes displaying medians, 25th and 75th percentiles, and whiskers extending to
1.5 of interquartile range. d Number of representative results (cryobiopsy vs. gastric biopsy forceps, representative
yes in red and no in blue). Representativity was assessed qualitatively by two expert
pathologists. Representativity was defined as the presence of ample evaluable bile
duct epithelium to rule out malignancy or, in case of carcinoma, the presence of ample
tumor cells and possible invasive growth allowing a definite diagnosis of malignancy.
Table 2
Clinical data from patients II (distribution biopsy attempts).
|
Method
|
Cryobiopsy attempts successful/overall
|
Cholangioforceps biopsy attempts successful/overall
|
Gastric biopsy forceps biopsy attempts successful/overall
|
Total biopsy attempts per patient successful/overall
|
|
Patient
|
|
1
|
3/3
|
0/0
|
0/0
|
3/3
|
|
2
|
0/3
|
0/3
|
0/0
|
0/6
|
|
3
|
3/4
|
1/5
|
0/0
|
4/9
|
|
4
|
0/3
|
0/0
|
3/3
|
3/6
|
|
5
|
3/5
|
0/0
|
2/3
|
5/8
|
|
6
|
3/4
|
0/0
|
1/3
|
4/7
|
|
7
|
3/4
|
0/0
|
1/3
|
4/7
|
|
8
|
3/4
|
0/3
|
2/3
|
5/10
|
|
9
|
3/4
|
0/3
|
1/3
|
4/10
|
|
10
|
2/3
|
1/3
|
3/3
|
6/9
|
|
11
|
1/3
|
1/3
|
3/3
|
5/9
|
|
12
|
1/3
|
0/3
|
1/3
|
2/9
|
|
13
|
3/3
|
3/3
|
3/3
|
9/9
|
|
14
|
0/4
|
0/3
|
3/3
|
3/10
|
|
Total
|
28/50
|
6/29
|
23/33
|
57/112
|
In the subgroup with pathologically altered areas (bile duct with overt tumor infiltration),
biopsies could be obtained successfully in 50 % of patient (3/6) with cholangioscopic
forceps, 55.5 % (5/9) with gastric biopsy forceps, and 81.8 % with cryobiopsy (9/11)
with no significant difference between the groups.
Histopathological specimen size
Mean specimen area obtained by gastric biopsy forceps was 3.3 ± 5.1 mm2 and 5.6 ± 4.5 mm2 for cryobiopsy. The specimen area of cryobiopsy was significantly larger compared
to gastric biopsy forceps (P = 0.0059) ([Fig. 2b]). A representative image of the automated size measurement from cryobiopsy is shown
in Supplementary Fig. 2a.
Histological assessment
Mean histological assessment quality score was 3.29 ± 1.34 and 4.35 ± 1.20 for gastric
biopsy forceps and cryobiopsy, respectively (P = 0.016, [Fig. 2c]).
Representativity
There was a good agreement between the two pathologists’ evaluations (Agreement 87.5 %,
Cohen’s Kappa of 0.69). Three tissue samples were excluded from the above-mentioned
analysis of specimen size and histological assessability due to a lack of evaluable
bile duct tissue. These samples were counted as not representative in this comparison
in analogy to an intention to treat analysis. Of 19 obtained samples for gastric biopsy
forceps 12 (63 %) were evaluated as representative by both pathologists. Twenty-three
of 25 samples of cryobiopsy (92 %) were representative (P = 0.027; [Fig. 2d]). Supplementary Fig. 2b and Supplementary Fig. 2c show examples of histological sections for gastric biopsy forceps and cryobiopsy.
Discussion
IBS are challenging, therefore, different biopsy techniques are required to improve
sensitivity [1]
[2]
[11]. One of those could be cryobiopsy. Several studies have demonstrated that cryobiopsy
is diagnostically superior to conventional biopsy techniques such as forceps e. g.
for lung biopsy [12]
[13].
In our study, the average sample area was significantly larger for cryobiopsy although
this difference was less pronounced than in other reports with similar cryoprobe dimensions
[9]
[10]
[14]. One explanation could be that the gastric biopsy forceps used in the current study
has a far wider opening diameter compared to bronchoscopic or cholangioscopic forceps.
Furthermore, in our current study only comparatively low activation times of the cryobiopsy
were used as damage to the tissue had to be prevented for further processing. Higher
activation times would yield larger specimens, but on the other hand, if activation
time is too high, specimens could potentially be too large to be retrieved through
the working channel of the cholangioscope and hence would have to be extracted together
with the cholangioscope, as is routinely done during bronchoscopic procedures [15]. In addition, longer activation times could risk bile duct perforation and bleeding
complications during the procedure, although histological evaluation showed no signs
of severe damage to the subepithelial tissue while performing cryobiopsy. Based on
the design of this ex vivo feasibility study we are unable to draw conclusions about
complications during the diagnostic procedure. This has to be addressed in further
studies.
During the current study, the low success rate of cholangioscopic forceps biopsies
(20.7 %) was surprising; however, this limitation is in line with our own clinical
observations. The tiny cholangioscopic forceps grasps only a very limited amount of
tissue and often is unable to penetrate the firm surface of the bile duct and its
tight connective tissue. By trend, gastric biopsy forceps surpassed cryobiopsy in
tissue acquisition from healthy biliary epithelium. Likely, the serrated surface of
the gastric biopsy forceps assists in grabbing tissue from the firm surface of the
bile ducts in comparison to the plane surface of the cholangioscopic forceps or cryobiopsy.
Nevertheless, both, representativity and histological assessment were significantly
larger for cryobiopsy compared to gastric biopsy forceps, especially from malign lesions,
possibly due to the scattered surface area compared to unaltered tissue. However,
due to the low numbers of included patients with tumor infiltration (only three patients
were included in the subgroup of patients with pathologically altered areas) our conclusion
concerning tumor patients is only of observatory nature.
Low case numbers overall can be discussed as further limitation of this study. The
main target was to investigate general feasibility of cryobiopsy in the human bile
duct. Therefore, an intended number of 15 cases was defined as sufficient to draw
conclusions about feasibility. Also, as this was a single-center study, cases of patients
who underwent pancreaticoduodenectomy were limited.
All experiments were performed on an imposed bile duct after lengthwise incision and
fixation to allow for a more standardized comparison of histolopathological representativity.
Therefore, the overall clinical challenge of improving the diagnostic yield in IBS
was not specifically addressed by our current study. The different biopsy techniques
ideally have to be compared during cholangioscopy or fluoroscopic guided biopsy of
intrahepatic lesions in a real-life setting. The cryoprobe prototype fits with 1.1-mm
diameter in every working channel of current cholangioscopes and has already been
field-tested in subsequent tests after this study.
Here we postulate a possible advantage of cryobiopsy because prior studies have indicated
it can be applied frontally and tangentially with equal efficacy [12]. This could be beneficial especially in small bile ducts with lacking flexibility
during cholangioscopy/ERCP.
Conclusions
This first systematic study of cryobiopsy in the biliary tract shows that cryobiopsy
is feasible, obtains larger tissue samples, and has superior quality compared to forceps
biopsies. Cryobiopsy represents a novel and effective method for tissue extraction
in the bile ducts and can help to improve the common clinical dilemma of IBS. With
these promising results, an in vivo study is justified.
